Clonal amplification of nucleic acid template molecules has proven to be very useful for a number of technologies, particularly for nucleic acid sequencing technologies where clonal populations of substantially identical copies amplified from a single template are immobilized on to a solid phase surface and utilized in subsequent sequencing operations. For example, a solid phase surface may comprise an independent solid phase surface (generally referred to as a “bead” substrate although such substrates are not necessarily spherical) that may be disposed in wells. The solid phase surface may also comprise other planar or non-planar surfaces and are used for immobilization and sequencing of clonal populations of amplified nucleic acid template. Typically, a species of amplification primer is disposed on the outer surface of the solid phase which may include surface within pores or cavities (i.e. in the case of a porous bead substrate) or within defined areas of a substrate surface (i.e. in the case of planar substrates that comprise many areas or “features” for amplification of individual template species of various possible shapes and sizes) in order to allow for parallel processing of many different template species.
Thus in many cases there are spatial limitations on the amount of surface area available as primer attachment sites and thus by extension on the amount of amplification product than can be bound to the solid phase substrate. For example, using common PCR reaction conditions, the total amount of nucleic acid template that can be amplified is capped by the total number of primers attached to the surface leading to a maximum 1:1 ratio of amplified nucleic acid template to primers. This can be problematic for downstream applications such as sequencing because the degree of signal obtainable from the population is proportional to the number of copies available to generate the signal where small numbers of copies may not generate enough signal to distinguish from experimental or background noise sources.
One solution has been to implement what is generally referred to as a concatamer PCR approach that makes it possible to increase the ratio of amplified nucleic acid template to primers to greater than 1:1. In other words producing more amplified nucleic acid template than there are primer sites on the surface.
To date concatamer PCR has been achieved in a couple of ways. A first approach requires the initial templates to contain tandem repeats of the starting template sequence which allow for self-priming and amplification. A second approach employed includes amplifying initial PCR products with a restriction enzyme site engineered into both ends of the fragments which can be subsequently digested producing “sticky” ends which can then be ligated to form the initial tandem repeat templates. This template would then be used in the concatamer PCR reaction.
The embodiments of the invention as described herein replace the requirement of tandem copies in the initial template of the concatamer PCR by employing a concatamer primer in the reaction and enables solid surface amplification strategies that allow for sequencing of the DNA template or other uses.
A number of references are cited herein, the entire disclosures of which are incorporated herein, in their entirety, by reference for all purposes. Further, none of these references, regardless of how characterized above, is admitted as prior art to the invention of the subject matter claimed herein.